Protein aggregation resulting from stress, disease or mutation poses a major threat to all cells. Damage due to protein aggregation is limited and repaired by a cellular protein quality control network consisting of chaperones and proteases. The proposed research investigates one component of this protein quality control network, the small heat shock proteins (sHSPs), a ubiquitous class of molecular chaperones. Expression and/or mutation of sHSPs are linked to multiple diseases of protein misfolding, including neurodegenerative diseases, myopathies and cataract. The mechanism of sHSP chaperone action and interaction with substrates, therefore, has wide-ranging implications for understanding cellular stress and disease processes, but remains poorly defined. Furthermore, sHSPs are an excellent model system for investigating the importance of protein dynamics in protein-protein recognition. We hypothesize that secondary, tertiary and quaternary dynamics underlie the effectiveness of sHSPs in recognizing and binding diverse denaturing proteins. Aim 1 will use crosslinking and advanced mass spectrometry to define the nature of substrate sites bound by sHSPs and the organization of sHSP-substrate complexes formed in vitro. How the cellular environment impacts substrate recognition will then be tested in vivo in E. coli. Aim 2 will determine how conformational flexibility of the sHSP N-terminal arm effects efficiency of sHSP substrate protection using iterations of molecular dynamics simulations and in vitro assays of the activity of mutant sHSPs. NMR will be used to obtain amino acid resolution of the dynamic interactions between sHSP and substrate. These complementary approaches will allow cross validation of results. sHSPs and substrates with known high resolution structures, or readily modeled by homology, will be used in Aims 1 and 2. The third and final Aim employs a model genetic system to test principles derived from the in vitro studies and to establish how sHSP-substrate interactions alter protein fate in the cell. In total, the proposed experiments will define not only how sHSPs recognize substrates and impact their metabolism, but also define new aspects of protein recognition and aggregate formation, which are critical to understanding many diseased states.